PB2507: EFFECTS OF TSH ON HEMOGLOBIN S POLYMERIZATION, DEFORMABILITY AND ADHESION ON SICKLE CELL ERYTHROCYTES
Authors: Evelyn Mendonça Reis, Lyzes Rosa Teixeira-Alves, Camila Cristina Guimarães-Nobre, Clemilson Berto-Junior
Journal: HemaSphere (2023)

Topic: 26. Sickle cell disease Background: Thyroid-stimulating hormone (TSH) is a peptide hormone synthesized and secreted by the pituitary gland and can affect the metabolism of the entire body and thyroid such as iodine uptake, production of thyroxine, and triiodothyronine. Its action is through the thyroid stimulating hormone receptor, a transmembrane G protein-coupled receptor, and it is already known that mature and enucleated erythrocytes express TSH receptors. Sickle Cell Disease (SCD) is a hereditary genetic disease that occurs when there is a homozygous exchange mutation of a glutamic acid residue for a valine residue in the beta-globin gene, leading to the formation of hemoglobin S (HbS). When it undergoes deoxygenation, it tends to polymerize, losing its biconcave shape and assuming a sickle shape. Patients with SCD have already been described to have metabolic and endocrine disorders, which can be the result of hypoxia, chronic anemia, iron overload, or genetic influence. Aims: Our work has as its main objective to study the relationship between TSH levels, the activation of its receptors in sickle erythrocytes, and the parameters which can be modulated through this relationship; for this, experiments were carried out to measure the HbS polymerization, deformability, and static adhesion. Methods: All experiments were carried out in triplicate and the TSH concentrations used were 1, 2, 3 and 5 mIU/L. The project was approved by the UFRJ Research Ethics Committee (Protocol 1032.889.952) and registered on the Brazil platform under the number CAAE 88140418.5.0000.5699. In the HbS polymerization assay, erythrocytes were placed in the presence of TSH concentrations (1,2,3 and 5 mIU/L), and then 2% sodium metabisulphite was added to stimulate polymerization, then the samples were read in a microplate reader for approximately 30 minutes at 1-minute intervals at 700nm. For the deformability experiment, a column with Sephacyl was prepared, where the samples were deposited and taken to a centrifuge at 3000 g and then analyzed in microplate reader at 540 nm and 700nm, where the analyzed samples were: control oxygenated containing erythrocytes and PBS, without the addition of metabisulfite; deoxygenated control, containing erythrocytes, PBS and metabisulfite; and erythrocytes with TSH (1,2,3 and 5 mIU/L) with PBS and metabisulfite. In the static adhesion test, thrombospondin was used to create a film in 96-well plates and verify erythrocyte adhesion in the presence of TSH (1,2,3 and 5 mIU/L). Thrombospondin was added to the plates and taken to a water bath for 1 hour, then 3% albumin was added for blocking and then the erythrocytes were added with TSH and after 2 hours in a water bath the excess was removed and 100 μL was added MiliQ water to the wells and then the samples were taken to the microplate reader for reading the plates at 540nm. Results: These results show that TSH at 1, 2, 3, and 5 mIU/L contractions were able to reduce polymerization of HbS and increase its deformability and that the static adhesion of erythrocytes at the lowest concentrations of 1 and 2 mIU/ L were increased, but at higher contractions of 3 and 5 mIU/L, which are closer to TSH contraction in sickle cell patients, static adhesion did not have a significant result.Keywords: Erythrocyte, Sickle cell disease